Hla-binding peptide, precursor thereof, and dna fragment and recombinant vector coding for said hla-binding peptide

ABSTRACT

An HLA-binding peptide binding to an HLA-A type molecule is provided that includes one or more types of amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 52, and not less than 8 and not more than 11 amino acid residues. All of these amino acid sequences are amino acid sequences predicted to bind to a human HLA-A molecule using a prediction program employing an active learning experiment method shown in FIG.  1.

TECHNICAL FIELD

The present invention relates to HLA-binding peptides, precursors thereof, and DNA fragments and recombinant vectors coding for the HLA-binding peptides.

BACKGROUND ART

When infection with a virus such as an influenza virus occurs, a virus elimination reaction due to natural immunity proceeds, a specific immune response is subsequently induced, and a virus elimination reaction proceeds.

In the specific immune response, virus in a body fluid is eliminated by a neutralizing antibody, and virus within a cell is eliminated by a cytotoxic T lymphocyte (CTL). That is, the CTL specifically recognizes a virus antigen (CTL epitope) consisting of 8 to 11 amino acids presented in an HLA class I molecule on the surface of an infected cell, and eliminates the virus by damaging the infected cell. Identifying such a virus-specific CTL epitope is therefore important for preparing preventive and therapeutic vaccines for the virus.

A technique of this kind is known from Patent Publication 1. Patent Publication 1 states that an oligopeptide formed from a specific amino acid sequence has the property of binding to an HLA.

-   [Patent Publication 1] Japanese Patent Application Laid-open No.     H8-151396 (1996)

DISCLOSURE OF THE INVENTION

However, the conventional technique described in the above-mentioned publication has room for improvement with regard to the following points.

Firstly, it is unclear whether or not the HLA-binding peptide of the above-mentioned publication binds to an HLA molecule effectively, and there is still room for improvement in terms of the HLA-binding properties.

Secondly, it is stated that the HLA-binding peptide of the above-mentioned publication has the property of binding to HLA-DQ4. However, it is unclear whether or not it binds to an HLA-A2 molecule (product of the HLA-A*0201 gene, HLA-A*0206 gene and the like), which is often seen in European and American people, and an HLA-A24 molecule (product of the HLA-A*2402 gene and the like), which is often seen in Japanese people.

The present invention has been accomplished under the above-mentioned circumstances, and provides an HLA-binding peptide that has excellent properties in binding to a specific type of HLA molecule.

According to the present invention, there is provided an HLA-binding peptide binding to an HLA-A type molecule, the HLA-binding peptide containing one or more types of amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 52, and consisting of not less than 8 and not more than 11 amino acid residues.

Furthermore, according to the present invention, there is provided the HLA-binding peptide, wherein it contains one or more types of amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 34, 35, 36, 37, 38, 40, 41, 43, 45, 47, 48, 49, 50, 51, and 52.

Moreover, according to the present invention, there is provided an HLA-binding peptide binding to an HLA-A type molecule, the HLA-binding peptide containing an amino acid sequence formed by deletion, substitution, or addition of one or two amino acid residues of the amino acid sequence contained in the above-mentioned HLA-binding peptide, and consisting of not less than 8 and not more than 11 amino acid residues.

In this way, the construct containing an amino acid sequence formed by deletion, substitution, or addition of one or a few amino acid residues of a specific amino acid sequence that has the property of binding to an HLA-A type molecule can also exhibit a similar effect to that of the above-mentioned HLA-binding peptide.

Furthermore, according to the present invention, there is provided a DNA fragment containing a DNA sequence coding for the above-mentioned HLA-binding peptide.

Moreover, according to the present invention, there is provided a recombinant vector containing a DNA sequence coding for the above-mentioned HLA-binding peptide.

Furthermore, according to the present invention, there is provided an HLA-binding peptide precursor changing within a mammalian body into the above-mentioned HLA-binding peptide.

In accordance with the present invention, since it contains a specific amino acid sequence, an HLA-binding peptide that has excellent properties in binding to an HLA-A type molecule can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned object, other objects, features, and advantages will become more apparent from preferred embodiments explained below by reference to the attached drawing.

[FIG. 1] A schematic drawing for explaining an active learning experiment design used in an embodiment.

BEST MODE FOR CARRYING OUT THE INVENTION

Modes for carrying out the present invention are explained below by reference to a drawing. In the drawing, similar components are denoted by similar reference numerals and symbols, and duplication of explanation is avoided as appropriate.

Embodiment 1

In this embodiment a peptide that contains an amino acid sequence for which the binding to an HLA molecule, predicted by a hypothesis obtained using an active learning experiment method (Japanese Patent Application Laid-open No. H11-316754 (1999)), is 3 or greater in terms of a -log Kd value, and consists of not less than 8 and not more than 11 amino acid residues is used as a candidate for an HLA-binding peptide. From the results of carrying out a binding experiment, it has been confirmed that these peptides are actually HLA-binding peptides.

As a result, a large number of HLA-binding peptides that have excellent properties in binding to an HLA-A type molecule because they contain amino acid sequence for which the binding to the HLA molecule in terms of a -log Kd value is 3 or greater could be obtained efficiently.

Specifically, the HLA-binding peptide related to this embodiment is an HLA-binding peptide that binds to an HLA-A type molecule, contains one or more types of amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 52, which will be described later, and consists of not less than 8 and not more than 11 amino acid residues.

Among human HLA-A types, about 50% of Japanese people have the HLA-A24 type. Many European and American people, such as German people, have the HLA-A2 type.

All of these sequences are sequences consisting of 9 amino acid residues contained in a certain genome protein of an avian influenza virus.

The sequences of SEQ ID NOS: 1 to 20 are given in Table 1 below.

TABLE 1 HLA-A24-binding peptides Binding SEQ ID Predicted SEQ Experiment No SEQ Score Name Data  1 WMACHSAAF 6.1873 330 7.27004  2 RLLQNSQVF 6.0988 305 6.91461  3 RLIQNSITI 5.9077  55 7.0483  4 IFLARSALI 5.8166 257 8.01161  S GQISVQPTF 5.7493 404 7.19131  6 ATNPIVPSF 5.6519   5-471 7.56449  7 ATNPVVPSF 5.6282 471 7.80781  8 NLPFERATI 5.5547 417 7.76375  9 ATSPIVPSF 5.5244   9-471 7.80229 10 IYRRRDGKW 5.514  5-96 7.49653 11 SLPFERATI 5.4943   9-417 7.71879 12 VGIDPFRLL 5.3829 299 5.12018 13 IYKRREGKW 5.3618  9-96 7.25015 14 RWVGGIGRF 5.3166  31 7.54336 15 RMVSGIGRF 5.2164  5-31 7.43594 16 DMSNEGSYF 5.1901 480 5.74415 17 IYKRRDGKW 5.1812  96 7.32598 18 DMNNEGSYF 5.169   5-480 5.37438 19 AEIEDLIFL 5.1369 251 20 IERMVLSAF 5.0612  63

The sequences of SEQ ID NOS: 1 to 20 are sequences consisting of 9 amino acid residues contained in a nucleoprotein of M22344 (H7) strain, AF508607 (H9) strain, or AY676037 (H5) strain, which are 3 representative serotypes (H7, H9, H5) of an avian influenza virus, which is described later. The sequences of SEQ ID NOS: 1 to 20 are sequences predicted by the above-mentioned method to be the highest in terms of binding to an HLA-A24 molecule (a product of the HLA-A*2402 gene). SEQ ID NOS: 1 to 20 are arranged in decreasing binding order. That is, SEQ ID NO: 1 is the sequence that is predicted to have the best binding. A predicted score for binding to the HLA-A24 molecule and binding experiment data for each sequence are expressed in the form of -log Kd values.

The sequences of SEQ ID NOS: 21 to 36 are given in Table 2 below.

TABLE 2 HLA-A2-binding peptides Binding SEQ ID Predicted SEQ Experiment No SEQ Score Name Data 21 YLEEHPSAG 5.3104  78 5.08483 22 SLPFERATI 5.3061   9-417 5.24328 23 AVKGVGTMV 5.083 182 5.57857 24 FRLLQNSQV 5.0517 304 4.45468 25 NLPFERATI 5.0017 417 26 YLEENFSAG 4.9503  9-78 4.90353 27 AVKGIGTMV 4.9476   9-182 4.8085 28 RLIQNSITI 4.9311  55 5.0127 29 SSFIRGTRV 4.9002 344 30 WMACHSAAF 4.8588 330 5.7894 31 FLARSALIL 4.8472 258 5.4765 32 CLPACVYGL 4.8118 275 33 SALILRGSV 4.7469 262 34 AQRAMMDQV 4.5751 234 5.33481 35 IFLARSALI 4.5056 257 5.93818 36 NATEIRASV 4.4764  21 4.74314

The sequences of SEQ ID NOS: 21 to 36 are sequences consisting of 9 amino acid residues contained in a nucleoprotein of M22344 (H7) strain, AF508607 (H9) strain, or AY676037 (H5) strain, which are 3 representative serum types (H7, H9, H5) of an avian influenza virus, which is described later. The sequences of SEQ ID NOS: 21 to 36 are sequences predicted by the above-mentioned method to be the highest in terms of binding to an HLA-A2 molecule (a product of the HLA-A*0201 gene). SEQ ID NOS: 21 to 36 are arranged in decreasing binding order. That is, SEQ ID NO: 21 is the sequence that is predicted to have the best binding. A predicted score for binding to the HLA-A2 molecule and binding experiment data for each sequence are expressed in the form of -log Kd values.

The sequences of SEQ ID NOS: 37 to 52 are given in Table 3 below.

TABLE 3 HLA-A2-binding peptides Binding SEQ ID Predicted SEQ Experiment No SEQ Score Name Data 37 SALILRGSV 5.4597 262 3.83934 38 AVKGVGTMV 5.3312 182 3.65413 39 MVLSAFDER 5.0975  66 40 AQRAMMDQV 5.0607 234 5.64316 41 AVKGIGTMV 5.0277   9-182 3.51984 42 ATIMAAFTG 4.9325 423 43 NATEIRASV 4.9117  21 5.70368 44 RTSDMRTEI 4.8958 436 45 RLIQNSITI 4.8951  55 4.42539 46 AAGAAVKGV 4.8858 178 47 FRLLQNSQV 4.7792 304 4.21314 48 FQGRGVFEL 4.6325 458 6.77438 49 LQNSQVFSL 4.5655 307 5.78131 50 FLARSALIL 4.4298 258 4.34141 51 LILYDKEEI 4.3648 108 5.38215 52 LIFLARSAL 4.3468 256 3.73085

The sequences of SEQ ID NOS: 37 to 52 are sequences consisting of 9 amino acid residues contained in a nucleoprotein of M22344 (H7) strain, AF508607 (H9) strain, or AY676037 (H5) strain, which are 3 representative serum types (H7, H9, H5) of an avian influenza virus, which is described later. The sequences of SEQ ID NOS: 37 to 52 are sequences predicted by the above-mentioned method to be the highest in terms of binding to an HLA-A2 molecule (a product of the HLA-A*0206 gene). SEQ ID NOS: 37 to 52 are arranged in decreasing binding order. That is, SEQ ID NO: 37 is the sequence that is predicted to have the best binding. A predicted score for binding to the HLA-A2 molecule and binding experiment data for each sequence are expressed in the form of -log Kd values.

Although details are described later, it is clear that in all of Tables 1 to 3 there is a correlation between the predicted score and the binding experiment data. That is, although there are slight errors, it can be said that a peptide that is predicted by the above-mentioned method to have high binding to the HLA-A molecule is found experimentally to have high binding to the HLA-A molecule.

Since there is no conventional technique for discovering an HLA-binding peptide by utilizing such an experimental design method, there are only a very small number of HLA-binding peptides that have been experimentally confirmed to have HLA-binding properties. Because of this, even when a peptide consisting of 9 amino acid residues is randomly synthesized by a conventional method and subjected to an experiment to find out if it binds to an HLA molecule, there is a probability of only about 1 in 100 of finding one that has a binding, in terms of a -log Kd value, exceeding 6.

In accordance with this embodiment, since the technique of finding an HLA-binding peptide by utilizing the experimental design method is used, as described above, as many as 52 sequences of HLA-binding peptides can be found. Furthermore, when the binding of some of the HLA-binding peptides obtained is experimentally examined, it is confirmed that all of the sequences that have been subjected to the experiment exhibit an excellent binding to HLA that is equal to or higher than that predicted.

Among these sequences, an HLA-binding peptide containing one or more types of amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 34, 35, 36, 37, 38, 40, 41, 43, 45, 47, 48, 49, 50, 51, and 52 is experimentally confirmed to bind to a human HLA-A type molecule. It can therefore be said with certainty that it is an HLA-binding peptide that has excellent properties in binding to a human HLA-A type molecule.

The binding to an HLA molecule of the HLA-binding peptide related to the present embodiment is 3 or greater in terms of a -log Kd value, particularly preferably 5 or greater, and more preferably 5.4 or greater.

In the field of biochemistry, it is known that a binding ability, in terms of a -log Kd value, of about 3 is the threshold level for whether or not a peptide actually binds to an MHC, which includes an HLA. Therefore, if the binding to an HLA molecule, in terms of a -log Kd value, is 3 or greater, it can be said that it is an HLA-binding peptide.

Furthermore, in the case of an HLA-A24 molecule, if the binding to the HLA-A24 molecule, in terms of a -log Kd value, is 5 or greater, since the peptide obtained has excellent properties in binding to the HLA molecule, it can suitably be used for the development of an effective therapeutic drug, prophylactic drug, and the like for an immune disease and the like.

Moreover, if the binding to an HLA-A24 molecule, in terms of a -log Kd value, is 5.4 or greater, the peptide obtained has particularly good properties in binding to the HLA molecule, and it can suitably be used for the development of an even more effective therapeutic drug, prophylactic drug, and the like for an immune disease and the like.

Furthermore, it may be arranged that the HLA-binding peptide related to the present embodiment consists of not less than 8 and not more than 11 amino acid residues.

In this way, if the peptide consists of not less than 8 and not more than 11 amino acid residues, it has excellent properties in binding to an HLA molecule. Furthermore, the cytotoxic T lymphocyte (CTL) specifically recognizes a virus antigen (CTL epitope) consisting of 8 to 11 amino acids presented in an HLA class I molecule on the surface of a cell infected with a virus and the like, and eliminates the virus by damaging the infected cell. It is important to prepare such a CTL epitope consisting of 8 to 11 amino acids that is specific to a virus and the like in order to prepare a vaccine for therapy or prevention against the virus and the like.

For example, the above-mentioned HLA-binding peptide may be a peptide consisting of amino acid residues alone, but it is not particularly limited thereto. For example, it may be an HLA-binding peptide precursor that is optionally modified with a sugar chain or a fatty acid group and the like as long as the effects of the present invention are not impaired. Such a precursor is subjected to a change involving digestion by a proteolytic enzyme and the like in a living mammalian body such as in a human digestive organ to become an HLA-binding peptide, thus exhibiting similar effects to those shown by the above-mentioned HLA-binding peptide.

Furthermore, the above-mentioned HLA-binding peptide may be a peptide that binds to a human HLA-A24 molecule.

Moreover, the above-mentioned HLA-binding peptide may also be a peptide that binds to a human HLA-A2 molecule.

In accordance with this constitution, since a peptide is obtained that binds to an HLA-A24 molecule, which is often seen in Asian people, such as Japanese people, it can be utilized in the development of a therapeutic drug, a prophylactic drug, and the like that is particularly effective for Asian people, such as Japanese people.

Furthermore, in accordance with this constitution also, since a peptide is obtained that binds to an HLA-A2 molecule, which is often seen in European and American people in addition to Japanese people, it can be utilized in the development of a therapeutic drug, a prophylactic drug, and the like that is particularly effective for European and American people in addition to Japanese people.

Furthermore, the amino acid sequence contained in the HLA-binding peptide may be an amino acid sequence derived from a certain genome protein of an avian influenza virus, but is not particularly limited. For example, it may be an amino acid sequence derived from an HIV protein, an amino acid sequence derived from a cedar pollen protein, and the like. It may also contain an amino acid sequence derived from another pathogenic or allergenic protein.

For example, when an amino acid sequence is contained that is derived from a nucleoprotein of an avian influenza virus, which is described later, an HLA-binding peptide that can be utilized in the prevention, treatment, and the like of a disease caused by the avian influenza virus can be obtained.

Embodiment 2

In accordance with this embodiment, there is provided an HLA-binding peptide that binds to an HLA-A type molecule, contains an amino acid sequence formed by deletion, substitution, or addition of one or two amino acid residues of the amino acid sequence contained in the above-mentioned HLA-binding peptide, and consists of not less than 8 and not more than 11 amino acid residues.

As described later, even though the constitution includes an amino acid sequence formed by deletion, substitution, or addition of one or a few amino acid residues of a specific amino acid sequence that binds to an HLA-A type molecule, similar effects to those of the HLA-binding peptide related to the above-mentioned embodiment 1 are exhibited.

The amino acid sequences of the nucleoproteins of M22344 strain, AF508607 strain, and AY676037 strain of the avian influenza virus are different from each other in part, but since the correlation between prediction data and experimental data for the -log Kd value is high, that is, a sequence that is determined from prediction data to have binding properties shows a good -log Kd value in experimental data, it can be predicted that even an amino acid sequence that is formed by deletion, substitution, or addition of one or two amino acid residues of an amino acid sequence that shows binding properties will show excellent HLA-binding properties in a similar manner.

Furthermore, it can be predicted that even an amino acid sequence formed by deletion, substitution, or addition of one or two amino acid residues of an amino acid sequence shown in SEQ ID NOS: 1 to 52 that has excellent properties in binding to an HLA-A molecule will show excellent HLA-binding properties in a similar manner.

From another viewpoint, it can be predicted that even an amino acid sequence formed by deletion, substitution, or addition of one or a few amino acid residues of an amino acid sequence predicted by the above-mentioned method to have excellent properties in binding to an HLA-A molecule will show excellent HLA-binding properties in a similar manner. The amino acid residues that are substituted are preferably amino acid residues having similar properties to each other, such as both being hydrophobic amino acid residues.

Moreover, the HLA-binding peptides described in Embodiment 1 and Embodiment 2 can be produced using a method known to a person skilled in the art. For example, they may be artificially synthesized by a solid-phase method or a liquid-phase method. Alternatively, these HLA-binding peptides may be produced by expressing them from a DNA fragment or a recombinant vector coding for these HLA-binding peptides. These HLA-binding peptides thus obtained can be identified by a method known to a person skilled in the art. For example, identification is possible by use of Edman degradation, mass spectrometry, and the like.

Embodiment 3

In accordance with the present embodiment, there is provided a DNA fragment containing a DNA sequence coding for the above-mentioned HLA-binding peptide. Since the DNA fragment related to the present embodiment contains a specific DNA sequence, it can express the above-mentioned HLA-binding peptide.

When the above-mentioned HLA-binding peptide is expressed by using the DNA fragment related to the present embodiment, expression may be carried out by incorporating this DNA fragment into a cell, or expression may be carried out by using a commercial artificial protein expression kit.

Furthermore, continuous expression may be carried out by incorporating the above-mentioned DNA fragment into, for example, a human cell. Because of this, an HLA-binding peptide can be made to be present continuously within a cell by incorporating a DNA fragment coding for the HLA-binding peptide into the cell rather than incorporating the HLA-binding peptide itself into the cell. When an HLA-binding peptide is used as a vaccine, such an ability to express continuously is advantageous in terms of enhancing the efficacy of the vaccine.

Moreover, the DNA fragment related to the present embodiment can be produced by a method known to a person skilled in the art. For example, it may be artificially synthesized by means of a commercial DNA synthesizer and the like. Alternatively, it may be segmented from the HCV genome by using a restriction enzyme and the like. Alternatively, it may be amplified from the HCV genome by a PCR method using a primer. The DNA fragment thus obtained may be identified using a method known to a person skilled in the art. For example, it may be identified by a commercial DNA sequencer.

Embodiment 4

In accordance with the present embodiment, there is provided a recombinant vector that contains a DNA sequence coding for the above-mentioned HLA-binding peptide. Since the recombinant vector related to the present embodiment contains a specific DNA sequence, the above-mentioned HLA-binding peptide can be expressed.

When the above-mentioned HLA-binding peptide is expressed by using the recombinant vector related to the present embodiment, expression may be carried out by incorporating this recombinant vector into a cell, or expression may be carried out by using a commercial artificial protein expression kit.

Furthermore, continuous expression may be carried out by incorporating the above-mentioned recombinant vector into, for example, a human cell. Because of this, the HLA-binding peptide can be made to be present continuously within a cell by incorporating a recombinant vector coding for the HLA-binding peptide into the cell rather than incorporating the HLA-binding peptide itself into the cell. When the HLA-binding peptide is used as a vaccine, such an ability to express continuously is advantageous in terms of enhancing the efficacy of the vaccine.

Furthermore, in the above-mentioned recombinant vector, the amount of HLA-binding peptide expressed can be controlled with high precision by the use of a certain sequence in a regulatory region involved in transcription and expression, such as a promoter region upstream of a DNA sequence coding for the above-mentioned HLA-binding peptide. Moreover, the copy number of the recombinant vector in a cell can be controlled with high precision by the use of a certain sequence in a regulatory region involved in replication, such as the origin region of the recombinant vector.

Furthermore, the above-mentioned recombinant vector may freely contain a sequence other than the DNA sequence coding for the above-mentioned HLA-binding peptide. For example, it may contain a sequence of a marker gene such as a drug resistance gene.

Moreover, the recombinant vector related to the present embodiment can be produced using a method known to a person skilled in the art. For example, it may be obtained by cleaving a multicloning site of a commercial vector such as pBR322 or pUC19 at a certain restriction enzyme site, and inserting the above-mentioned DNA fragment into the site and carrying out ligation. Furthermore, the recombinant vector thus obtained can be identified using a method known to a person skilled in the art. For example, it can be confirmed by agarose gel electrophoresis whether or not the length of the DNA fragment cleaved by a predetermined restriction enzyme coincides with the restriction map of a commercial vector such as pBR322 or pUC19 and, furthermore, it can be identified by a DNA sequencer and the like whether or not the above-mentioned DNA sequence is contained in the DNA sequence cut out from the multicloning site.

Embodiments of the present invention are described above, but they are exemplifications of the present invention, and various constitutions other than those above may be employed.

For example, in the embodiments above, an HLA-binding peptide containing an amino acid sequence derived from a certain genome protein of avian influenza virus is used, but an HLA-binding peptide containing an amino acid sequence derived from another protein of avian influenza virus may be used. In such a case, it can be utilized in the treatment of various immune diseases related to the protein from which it is derived.

Furthermore, it may be an HLA-binding peptide for a pathogen other than avian influenza virus, such as an HIV virus, or an allergen such as cedar pollen, or an HLA-binding peptide containing an amino acid sequence derived from a protein such as a cancer cell.

It can be anticipated that if an amino sequence is contained that is predicted using the above-mentioned method to have excellent binding to HLA, it will shown excellent binding properties to HLA in a similar manner when it is experimentally confirmed. Because of this, these HLA-binding peptides can be used suitably in treatment or prevention centering around infectious diseases (influenza, SARS, HIV, HCV, and the like), and in cancer immunotherapy, allergic disease (hay fever, rheumatism, atopy, asthma, and the like), autoimmune disease, and the like.

EXAMPLES

The present invention is further explained below by reference to Examples, but the present invention is not limited thereto.

Specifically, procedures of prediction, experiment, and evaluation in the present examples were carried out based on an active learning experiment design, and in general the following steps were repeated. A schematic drawing for the active learning experiment design employed here is shown in FIG. 1.

(1) A trial of a lower-order learning algorithm, which will be described later, was carried out once. That is, a plurality of hypotheses were generated by random sampling from accumulated data and, with regard to randomly expressed candidate query points (peptides), a point that showed the largest distribution of predicted values was selected as a query point to be subjected to an experiment.

(2) The peptide at the selected query point was prepared by a synthesis and purification method, which will be described later, and the actual binding ability was measured by an experiment, which will be described later, and added to accumulated data.

In the present example, as the lower-order learning algorithm, a supervised learning algorithm of a Hidden Markov Model was used, and 20 to 30 types of peptides were predicted and selected per experiment by starting with the initial data for 223 types of peptides; the above-mentioned procedure was repeated four times, and a total of 341 data points were obtained.

More specifically, in the active learning method of the present example, 20 to 30 types of peptides containing an amino acid sequence in which 9 of 20 types of amino acids were arranged were designed and synthesized per experiment. The strength of binding (binding ability) thereof to an HLA molecule was measured. The binding ability (Kd value) was obtained as an experimental result. When the binding ability was high, the peptide was selected as a candidate for an HLA-binding peptide that could be used as a material for a vaccine.

The results thus obtained were inputted into a learning system equipped with a learning machine employing the Hidden Markov Model as a mathematical algorithm, and rules were created. The learning machine sampled different results to prepare the rules. The rules expressed by the learning machine had different constitutions. The rules thus obtained and experimental data were stored as needed as accumulated data.

From among more than 20⁹=500 billion peptide sequences, candidates for a subsequent experiment were selected by the rules, and the above-mentioned process was repeated. In this stage, different rules were applied to experimental candidates, and the candidates for which predictions of the experimental results were divided were subjected to experiment. In this way, since the candidates for which predictions of the experimental results were divided were subjected to subsequent experiment, the final precision of the prediction was increased.

In this way, a plurality of learning machines carried out selective sampling in which samples that would give different predictions were selected as experimental candidates, information could be gained efficiently, and a hypothesis (rule) with high precision could be obtained. Repeating the above-mentioned process four times gave excellent results as in Examples described later. Repeating it seven times or more gave even better results.

In accordance with such an active learning method, the number of repetitions of the binding experiment for peptides consisting of 9 amino acid residues, which would otherwise have to be carried out for the 500 billion or more combinations of all the candidates for HLA-binding peptides, could be reduced. In the active learning method, a rule was formed by experiment, and the experiment was repeated for tens of sequence candidates that were predicted by applying the rule. Because of this, the number of experiments could be cut, and the time and cost of the initial screening could be greatly reduced.

Furthermore, the hit rate for prediction of the binding of a peptide to HLA by the rule obtained by the active learning method reached 70 to 80%, whereas the hit rate by other known techniques such as the anchor method was as low as about 30%.

<Synthesis and Purification of Peptide>

A peptide was manually synthesized by the Merrifield solid-phase method using Fmoc amino acids. After deprotection, reverse phase HPLC purification was carried out using a C18 column to give a purity of 95% or higher. Identification of the peptide and confirmation of its purity were carried out using a MALDI-TOF mass spectrometer (Voyager DE RP, PerSeptive). Quantitative analysis of the peptide was carried out by a Micro BCA assay (Pierce Corp.) using BSA as a standard protein.

<Experiment of Binding Peptide to HLA-A2402 Molecule>

The ability of a peptide to bind to an HLA-A24 molecule, which is a product of the HLA-A*2402 gene, was measured using (ClR-A24 cells expressing the HLA-A24 gene (cells produced by Professor Masafumi Takiguchi, Kumamoto University being supplied with permission).

ClR-A24 cells were first exposed to acidic conditions at a pH of 3.3 for 30 seconds, thus dissociating and removing a light chain β2m, which is associated with HLA class I molecules in common, and an endogenous peptide originally bound to the HLA-A*2402 molecule. After neutralization, purified β2m was added to ClR-A24 cells, the obtained product was added to serial dilutions of a peptide, and incubated on ice for 4 hours. Staining was carried out using fluorescently labeled monoclonal antibody 17A12, which recognizes association (MHC-pep) of the three members, that is, HLA-A*2402 molecule, the peptide, and β2m, which had reassociated during the incubation.

Subsequently, the MHC-pep count per ClR-A24 cell (proportional to the strength of fluorescence of the above-mentioned fluorescent antibody) was quantitatively measured using a FACScan fluorescence-activated cell sorter (Becton Dickinson Biosciences). A binding dissociation constant Kd value between the HLA-A24 molecule and the peptide was calculated from the average strength of fluorescence per cell by a published method (Udaka et al., Immunogenetics, 51, 816-828, 2000).

<Experiment of Binding Peptide to HLA-A0201 Molecule>

The ability of a peptide to bind to an HLA-A2 molecule, which is a product of the HLA-A*0201 gene, was measured using strain JY cells (obtained from ATCC (American Type Culture Collection)) expressing the HLA-A*0201.

JY cells were first exposed to acidic conditions at a pH of 3.8 for 30 seconds, thus dissociating and removing a light chain β2m and an endogenous peptide, which were noncovalently associated with the HLA-A*0201 molecule. After neutralization, a reassociation experiment was carried out.

The above-mentioned JY cells and the purified β2m were added to stepped dilutions of peptide for which the binding ability would be measured, and incubation was carried out on ice for 4 hours. HLA-A*0201 molecules that had reassociated up to this point were stained using the associating type specific fluorescently-labeled monoclonal antibody BB7.2.

Subsequently, the amount of fluorescence per cell was measured using a flow cytometer and a dissociation constant Kd value was calculated by a published method (Udaka et al., Immunogenetics, 51, 816-828, 2000).

<Experiment of Binding Peptide to HLA-A0206 Molecule>

The ability of a peptide to bind to an HLA-A2 molecule, which is a product of the HLA-A*0206 gene, was measured using RA2.6 cells (cell strain newly prepared in Kochi University) in which cDNA of the HLA-A*0206 gene is expressed in RAMS cells, which are mouse TAP peptide transporter deficient cells.

RA2.6 cells were first cultured overnight at 26° C.; when HLA-A*0206 molecules having no peptide bound thereto were deposited on the cell surface, stepped dilutions of peptide were added; binding was carried out at room temperature for 30 minutes.

Subsequently, culturing was carried out at 37° C. for 3.5 hours, empty HLA-A*0206 molecules to which no peptide was bound were denatured, and the tertiary structure was lost.

The cells were stained by adding thereto fluorescently labeled monoclonal antibody 17A10 or 17A12, which specifically recognize the peptide-binding HLA-A*0206 molecule, and incubating on ice for 20 minutes.

Subsequently, the amount of fluorescence per cell was measured using a flow cytometer, and a dissociation constant Kd value was calculated by a published method (Udaka et al., Immunogenetics, 51, 816-828, 2000).

<Evaluation Results>

The prediction results and the experimental results shown in Table 1 to Table 3 above were obtained.

The sequences of SEQ ID NOS: 1 to 20 in Table 1 are sequences consisting of 9 amino acid residues contained in the full-length sequence of a nucleoprotein of M22344 strain, AF508607 strain, or AY676037 strain of avian influenza virus registered in GENBANK. The sequences of SEQ ID NOS: 1 to 20 are sequences predicted by a hypothesis obtained using the experimental design method explained in Embodiment 1 to be the highest in terms of binding to an HLA-A24 molecule (a product of the HLA-A*2402 gene). SEQ ID NOS: 1 to 20 are arranged in decreasing binding order. That is, SEQ ID NO: 1 is the sequence that is predicted to have the best binding. The full-length amino acid sequence of the nucleoprotein of M22344 strain of avian influenza virus is shown in SEQ ID NO: 53

(MASQGTKRSYEQMETGGERQNATEIRASVGRMVGGIGRFYIQMCTELKLSDYEGRLI QNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYKRRDGKWMRELILYDKEE IRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLP RRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQ TAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGY DFEREGYSLVGIDPFRLLQNSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFI RGTRVVPRGQLSTRGVQIASNENMETMDSSTLELRSRYWAIRTRSGGNTNQQRASAGQ ISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGV FELSDEKATNPVVPSFDMSNEGSYFFGDNAEEYDN), the full-length amino acid sequence of the nucleoprotein of AF508607 strain is shown in SEQ ID NO: 54 (MALQGTKRSYEQMETGGERQNATEIRASVGRMVGGIGRFYIQMCTELKLSDHEGRLI QNSITIERMVLSAFDERRNRYLEENPSAGKDPKKTGGPIYKRREGKWVRELILYDKEE IRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLP RRSGAAGAAVKGIGTMVMELIRMIKRGINDRNFWRGDNGRRTRIAYERMCNILKGKFQ TEAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGY DFEREGYSLVGIDPFRLLQNSQVFSLIRSNENPAHKSQLVWMACHSAAFEDLRVSSFI RGTRVIPRGQLSTRGVQIASNENMETIDSSTLELRSRYWAIRTRSGGNTNQHRASAGQ ISVQPTFSVQRSLPFERATIMAAFTGNTEGRTSDMRTEIIRMMENAKPEDVSFQGRGV FELSDEKATSPIVPSFDMSNEGSYFFGDNAEEYD), and the full-length amino acid sequence of the nucleoprotein of AY676037 strain of avian influenza virus is shown in SEQ ID NO: 55 (MASQGTKRSYEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTELKLSDYEGRLI QNSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVRELILYDKEE IRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLP RRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQ TAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGY DFEREGYSLVGIDPFRLLQNSQVFSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFI RGTRVVPRGQLSTRGVQIASNENMEAMDSNTLELRSRYWAIRTRSGGNTNQQRASAGQ ISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGV FELSDEKATNPIVPSFDMNNEGSYFFGDNAEEYDN).

Furthermore, the sequences of SEQ ID NOS: 21 to 36 in Table 2 are sequences consisting of 9 amino acid residues contained in a nucleoprotein of M22344 strain, AF508607 strain, or AY676037 strain of the above-mentioned avian influenza virus. The sequences of SEQ ID NOS: 21 to 36 are sequences predicted by a hypothesis obtained using the experimental design method explained in Embodiment 1 to be the highest in terms of binding to an HLA-A2 molecule (a product of the HLA-A*0201 gene). SEQ ID NOS: 21 to 36 are arranged in decreasing binding order. That is, SEQ ID NO: 21 is the sequence that is predicted to have the best binding.

Moreover, the sequences of SEQ ID NOS: 37 to 52 in Table 3 are sequences consisting of 9 amino acid residues contained in a nucleoprotein of M22344 strain, AF508607 strain, or AY676037 strain of the above-mentioned avian influenza virus. The sequences of SEQ ID NOS: 37 to 52 are sequences predicted by a hypothesis obtained using the experimental design method explained in Embodiment 1 to be the highest in terms of binding to an HLA-A2 molecule (a product of the HLA-A*0206 gene). SEQ ID NOS: 37 to 52 are arranged in decreasing binding order. That is, SEQ ID NO: 37 is the sequence that is predicted to have the best binding.

Table 1 to Table 3 show, with regard to each of the nucleoproteins of M22344 strain, AF508607 strain, or AY676037 strain of avian influenza virus, the amino acid sequences with the highest scores in the predicted results obtained using the above-mentioned prediction program, the predicted score, and the corresponding binding experiment data. All of the binding experiments were obtained by artificially synthesizing a 9-amino acid peptide by the above-mentioned synthetic method.

Although the amino acid sequences of the nucleoproteins of avian influenza virus M22344 strain, AF508607 strain, and AY676037 strain are registered in GenBank, sequences consisting of 9 amino acid residues thereamong, which become HLA-binding peptides, are not currently registered.

There are a plurality of serum types for the avian influenza virus that have a possibility of infecting humans; among them M22344 strain (H7 type) is the type of influenza that is currently (as of November 2005) spreading mainly in Europe, and AY676037 strain (H5 type) is the type of influenza that is currently spreading mainly in Asian but also in Europe. In this example, an HLA-binding peptide contained in the nucleoprotein of such an influenza virus epidemic strain, which is spreading in Europe or Asia, has been found. This HLA-binding peptide can suitably be utilized in the development of preventive/therapeutic vaccines for avian influenza in Europe and Asia.

Here, the amino acid sequences of the nucleoproteins of M22344 strain, AF508607 strain, and AY676037 strain of the avian influenza virus are different from each other in part, but it can be predicted that even amino acid sequences in which one or a few amino acid residues of the amino acid sequences are substituted for each other will show excellent HLA-binding properties in the same way as described above.

For example, the third from the left in the SEQ ID NO: 7 peptide of the M22344 strain is N, whereas in the SEQ ID NO: 9 peptide of the AF508607 strain it is S instead of N, and the fifth from the left in the SEQ ID NO: 7 peptide of the M22344 strain is V, whereas in the SEQ ID NO: 9 peptide of the AF508607 strain and the SEQ ID NO: 6 peptide of the AY676037 strain it is I instead of V.

Furthermore, for example, the first from the left in the SEQ ID NO: 8 peptide of the M22344 strain is N, whereas in the SEQ ID NO: 11 peptide of the AF508607 strain it is S instead of N.

Moreover, for example, the fourth from the left in the SEQ ID NO: 14 peptide of the M22344 strain is G, whereas in the SEQ ID NO: 15 peptide of the AY676037 strain it is S instead of G.

Furthermore, for example, the third from the left in the SEQ ID NO: 16 peptide of the M22344 strain is S, whereas in the SEQ ID NO: 18 peptide of the AY676037 strain it is N instead of S.

Moreover, for example, the sixth from the left in the SEQ ID NO: 17 peptide of the M22344 strain is D, whereas in the SEQ ID NO: 13 peptide of the AF508607 strain it is E instead of D, and the third from the left in the SEQ ID NO: 17 peptide of the M22344 strain is K, whereas in the SEQ ID NO: 10 peptide of the AY676037 strain it is R instead of K.

Furthermore, for example, the fifth from the left in the SEQ ID NO: 21 peptide of the M22344 strain is H, whereas in the SEQ ID NO: 26 peptide of the AF508607 strain it is N instead of H.

Moreover, for example, the fifth from the left in the SEQ ID NO: 23 peptide of the M22344 strain is V, whereas in the SEQ ID NO: 27 peptide of the AF508607 strain it is I instead of V.

Among the peptide sequences in which single amino acid residues are substituted for each other, for example, the third from the left in the SEQ ID NO: 7 peptide of the M22344 strain is N, whereas in the SEQ ID NO: 9 peptide of the AF508607 strain it is S instead of N, and the experimental binding value for the SEQ ID NO: 7 peptide of the M22344 strain is 7.80781, whereas the experimental binding value for the SEQ ID NO: 9 peptide of the AF508607 strain is 7.80229. Furthermore, the fifth from the left in the SEQ ID NO: 7 peptide of the M22344 strain is V, whereas in the SEQ ID NO: 9 peptide of the AF508607 strain and the SEQ ID NO: 6 peptide of the AY676037 strain it is I instead of V, and the experimental binding value for the SEQ ID NO: 7 peptide of the M22344 strain is 7.80781, whereas the experimental binding value for the SEQ ID NO: 9 peptide of the AF508607 strain is 7.80229 and the experimental binding value for the SEQ ID NO: 6 peptide of the AY676037 strain is 7.56449, thus confirming that binding is good in all cases.

Furthermore, among the peptide sequences in which single amino acid residues are substituted for each other, for example, the first from the left in the SEQ ID NO: 8 peptide of the M22344 strain is N, whereas in the SEQ ID NO: 11 peptide of the AF508607 strain it is S instead of N, and the experimental binding value for the SEQ ID NO: 8 peptide of the M22344 strain is 7.76375, whereas the experimental binding value for the SEQ ID NO: 11 peptide of the AF508607 strain is 7.71879, thus confirming that binding is good in either case.

Moreover, among the peptide sequences in which single amino acid residues are substituted for each other, for example, the fourth from the left in the SEQ ID NO: 14 peptide of the M22344 strain is G, whereas in the SEQ ID NO: 15 peptide of the AY676037 strain it is S instead of G, and the experimental binding value for the SEQ ID NO: 14 peptide of the M22344 strain is 7.54336, whereas the experimental binding value for the SEQ ID NO: 15 peptide of the AY676037 strain is 7.43594, thus confirming that binding is good in either case.

Furthermore, among the peptide sequences in which single amino acid residues are substituted for each other, for example, the third from the left in the SEQ ID NO: 16 peptide of the M22344 strain is S, whereas in the SEQ ID NO: 18 peptide of the AY676037 strain it is N instead of S, and the experimental binding value for the SEQ ID NO: 16 peptide of the M22344 strain is 5.74415, whereas the experimental binding value for the SEQ ID NO: 18 peptide of the AY676037 strain is 5.37438, thus confirming that binding is good in either case.

Moreover, among the peptide sequences in which single amino acid residues are substituted for each other, for example, the sixth from the left in the SEQ ID NO: 17 peptide of the M22344 strain is D, whereas in the SEQ ID NO: 13 peptide of the AF508607 strain it is E instead of D, and the experimental binding value for the SEQ ID NO: 17 peptide of the M22344 strain is 7.32598 whereas the experimental binding value for the SEQ ID NO: 13 peptide of the AF508607 strain is 7.25015. Furthermore, the third from the left in the SEQ ID NO: 17 peptide of the M22344 strain is K, whereas in the SEQ ID NO: 10 peptide of the AY676037 strain it is R instead of K, and the experimental binding value for the SEQ ID NO: 17 peptide of the M22344 strain is 7.32598, whereas the experimental binding value for the SEQ ID NO: 10 peptide of the AY676037 strain is 7.49653, thus confirming that binding is good in all cases.

Furthermore, among the peptide sequences in which single amino acid residues are substituted for each other, for example, the fifth from the left in the SEQ ID NO: 21 peptide of the M22344 strain is H, whereas in the SEQ ID NO: 26 peptide of the AF508607 strain it is N instead of H, and the experimental binding value for the SEQ ID NO: 21 peptide of the M22344 strain is 5.08483, whereas the experimental binding value for the SEQ ID NO: 26 peptide of the AF508607 strain is 4.90353, thus confirming that binding is good in either case.

Moreover, among the peptide sequences in which single amino acid residues are substituted for each other, for example, the fifth from the left in the SEQ ID NO: 23 peptide of the M22344 strain is V, whereas in the SEQ ID NO: 27 peptide of the AF508607 strain it is I instead of V, and the experimental binding value for the SEQ ID NO: 23 peptide of the M22344 strain is 5.57857 whereas the experimental binding value for the SEQ ID NO: 27 peptide of the AF508607 strain is 4.8085, thus confirming that binding is good in either case.

It can therefore be predicted that any of the peptide sequences in which one or two amino acid residues are substituted for each other will show excellent binding to an HLA-A molecule. In conclusion, even an amino acid sequence formed by deletion, substitution, or addition of one or a few amino acid residues of an amino acid sequence shown by SEQ ID NOS: 1 to 52 that has excellent properties in binding to an HLA-A molecule can be predicted to similarly show excellent HLA-binding properties.

From another viewpoint, even an amino acid sequence formed by deletion, substitution, or addition of one or a few amino acid residues of an amino acid sequence that has excellent properties in binding to an HLA-A molecule as predicted by the hypothesis obtained by the experimental design method explained in Embodiment 1 similarly can be said to show excellent HLA-binding properties. The amino acid residues that are substituted are preferably amino acid residues that have similar properties to each other, such as the two being hydrophobic amino acid residues.

The present invention is explained above by reference to Examples. These Examples are only illustrated as examples, and a person skilled in the art will understand that various modification examples are possible, and such modification examples are included in the scope of the present invention.

For example, in the examples above, the nucleoprotein of the M22344 strain, AF508607 strain, or AY676037 strain of avian influenza virus was used, but another protein or another strain of the avian influenza virus may be used. In this case also, in accordance with the prediction program used in the present invention, HLA binding properties can be predicted with high accuracy. 

1. An HLA-binding peptide binding to an HLA-A type molecule, said HLA-binding peptide comprising one or more types of amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 52, and not less than 8 and not more than 11 amino acid residues.
 2. The HLA-binding peptide as set forth in claim 1, comprising one or more types of amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12,13, 14, 15, 16,17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 34, 35, 36, 37, 38, 40, 41, 43, 45, 47, 48, 49, 50, 51, and
 52. 3. An HLA-binding peptide binding to an HLA-A type molecule, said HLA-binding peptide comprising an amino acid sequence formed by deletion, substitution, or addition of one or two amino acid residues of said amino acid sequence contained in the HLA-binding peptide as set forth in claim 1, and not less than 8 and not more than 11 amino acid residues.
 4. The HLA-binding peptide as set forth in claim 1, wherein said HLA-binding peptide binds to a human HLA-A*2402 molecule.
 5. The HLA-binding peptide as set forth in claim 1, wherein said HLA-binding peptide binds to a human HLA-A*0201 molecule.
 6. The HLA-binding peptide as set forth in claim 1, wherein said HLA-binding peptide binds to a human HLA-A*0206 molecule.
 7. A DNA fragment comprising a DNA sequence coding for the HLA-binding peptide as set forth in claim
 1. 8. A recombinant vector comprising a DNA sequence coding for the HLA-binding peptide as set forth in claim
 1. 9. An HLA-binding peptide precursor changing within a mammalian body into the HLA-binding peptide as set forth in claim
 1. 